Kinetic and X-ray structural studies of a mutant Escherichia coli alkaline phosphatase (His-412-->Gln) at one of the zinc binding sites.
Kinetic and X-ray structural studies of a mutant Escherichia coli alkaline phosphatase (His-412-->Gln) at one of the zinc binding sites.
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对突变型大肠杆菌碱性磷酸酶 (His-412-->Gln) 在锌结合位点之一的动力学和 X 射线结构研究。
DOI：
10.1021/bi9523421
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Kantrowitz,ER
中科院分区：
文献类型：
--
作者：
Ma,L;Kantrowitz,ER
在大肠杆菌碱性磷酸酶中，用谷氨酰胺替代His-412已被应用于位点特异性诱变。在野生型酶中，His-412是活性位点上具有重要催化作用的锌原子（Zn1）的直接配体。突变酶（H412Q）表现出相同的kcat，但与野生型酶的相应动力学参数相比，inkm增加了50倍。此外，H412Q酶的锌含量低于野生型酶。与野生型酶相比，Tris在转移酶反应中作用较弱，并显著抑制H412Q酶的水解反应。添加锌后，突变酶的kcatvalue高于野生型酶，部分恢复了弱底物和磷酸盐结合，也减轻了Tris的抑制作用。H412Q酶的结构也通过x射线晶体学测定。H412Q酶的整体结构与野生型酶非常相似；突变位点附近只有α-碳位移超过1 Å。在H412Q结构中，酶的活性位点没有结合磷酸；然而，在磷酸通常与野生型酶结合的地方观察到两个水分子。对H412Q结构活性位点的仔细检查显示Ser-102和突变位点的结构变化。例如，Gln-412侧链的羰基氧在野生型结构中旋转远离His-412的位置，尽管距离太远（3.2 Å）而无法与Zn1协调。对H412Q酶的研究以及对H412Q和H412N结构的比较表明，组氨酸咪唑环的结构和静电对其在碱性磷酸酶中作为锌配体的功能至关重要。
Site-specific mutagenesis has been used to replace His-412 with glutamine inEscherichia colialkaline phosphatase. In the wild-type enzyme His-412 is a direct ligand to one of the catalytically important zinc atoms (Zn1) in the active site. The mutant enzyme (H412Q) exhibited about the samekcat, but a 50-fold increase inKmcompared to the corresponding kinetic parameters for the wild-type enzyme. Furthermore, the H412Q enzyme had a lower zinc content than the wild-type enzyme. In contrast to the wild-type enzyme, Tris was less effective in the transferase reaction and dramatically inhibited the hydrolysis reaction of the H412Q enzyme. The addition of zinc to the mutant enzyme increased thekcatvalue above that of the wild-type enzyme, partially restored the weak substrate and phosphate binding, and also alleviated the inhibition by Tris. The structure of the H412Q enzyme was also determined by X-ray crystallography. The overall structure of the H412Q enzyme was very similar to that of the wild-type enzyme; the only α-carbon displacements over 1 Å were observed near the mutation site. In the H412Q structure no phosphate was bound in the active site of the enzyme; however, two water molecules were observed where phosphate normally binds in the wild-type enzyme. Close examination of the active site of the H412Q structure revealed structural changes in Ser-102 as well as at the mutation site. For example, the carbonyl oxygen of the side chain of Gln-412 rotated away from the position of His-412 in the wild-type structure, although too far away (3.2 Å) to coordinate to Zn1. Studies on the H412Q enzyme, and a comparison of the H412Q and H412N structures, suggest that the structure and electrostatics of the imidazole ring of histidine are critical for its function as a zinc ligand in alkaline phosphatase.